Current Analytical Chemistry, 2006, 2, 00-00
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Single Cell Proteomics: Challenge for Current Analytical Science Bo Xu, Wei Du, Bi-Feng Liu* and Qingming Luo The Key Laboratory of Biomedical Photonics of Ministry of Education, and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Huazhong University of Science and Technology, Wuhan, 430074, P. R. China Abstract: At present, proteomics that promises deep understanding of genomic functions is developing rapidly. Proteomics at single cell level represents new challenges that greatly call for technique innovation in current analytical chemistry. In this mini-review, the concept of single cell proteomics (SCP) is clarified. Recent advancement on strategy and methodology of SCP is reviewed, with emphasis on micro-separation technique improvement such as ultra-thin layer gel electrophoresis, multidimensional micro liquid chromatography and capillary electrophoresis, and microfluidic chip as well. Future development on SCP is also suggested.
Keywords: Single cell proteomics, protein separation, chromatography, capillary electrophoresis, microfluidic chip, review. 1. INTRODUCTION 1.1 From Genomics to Proteomics After the completion of human genome project, it is recognized that the genes are merely the carriers of genetic information. The executors of life activities are the proteins that are the products of genes expression. In post-genomic era, the research interest is changing from the revelation of the genetic information of biological species to the systematically functional investigation [1-3]. With this shift, functional genomics that attempts to discover the function of genes at genomic level is developing rapidly, which strongly calls for technology innovations [4-9]. For example, highthroughput technologies like DNA microarray chips are well developed to detect global gene expression at transcriptomic level [10-13]. But it is far from enough. Generally, expression at mRNA level can not represent that at proteins level [14]. Even the presence of an open reading frame does not guarantee the existence of a protein. A single gene can encode multiple different proteins, with alternative splicing of the mRNA transcript and varying translation start or stop sites, or frame-shifting in which a different set of triplet codons in the mRNA is translated [15]. Further more, the intrinsic characteristics of proteins such as post-translational modification, transport localization, structure formation, metabolism, interaction among proteins and other biomolecules, cannot be learned from investigations at genomic level. All of these possibilities result in a proteome estimated to be an order of magnitude more complex than the genome [15]. What is more, proteome is a dynamic concept. It varies not only in different tissues and different cells of the same organism, but also in the different development stages of the same organism. And
it varies in different physiological conditions of the organism as well as in different external environment. Finally, a single protein may be involved in more than one process. Or similar functions may be carried out by various proteins. Therefore, the shift from genomics to proteomics is a difficult mission. The concept of “Proteome” is firstly proposed by Wilkins and Williams [16] in 1995. Proteomic strategies attempt to utilize the information from genome to find out protein function. As stated by Fields [15], “Proteomics includes not only the identification and quantification of proteins, but also the determination of their localizations, modifications, interactions, activities, and ultimately, their functions.” With the help of proteomics, we will solve the problem of spatialtemporal expression of proteins, which cannot be settled by genomics. For instance, proteome is heterogeneous and variable. The category and quantity of the proteins in different cells of the identical organism differ from each other. Even in homogeneous cells, their proteomes are in variation under different development stages and environmental conditions. Moreover, in the pathologic or therapeutic procedure, the expression level of cell proteome is different from normal state. Traditional research methods are usually static and local, generally taking individual gene or protein as research objects. Such methods would show great limitation when they are applied to complicated and dynamic life activities or diseases. Therefore, proteomic method is the right way to resolve these problems [17-19]. In viewpoint of systems biology, protein molecules need comprehensive and systematic understanding as the basic elements in the biological systems [20-22]. A primary task is to separate and identify proteins from cells, which is in great need of proteomic approaches. 1. 2 The Concept of Single Cell Proteomics (SCP)
*Address correspondence to this author at the The Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology (Eastern Campus), Wuhan 430074, P.R. China; Tel: +86-27-8746-4580; Fax: +86-27-8746-4570; E-mail:
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It is estimated that a typical mammalian cell of about 10 m m in diameter has a volume of about 500 femtoliters, and contains only about 50 picograms (2 femtomoles) of total proteins [23]. Such extremely trace quantity can not be handled by traditional methods. In conventional way, © 2006 Bentham Science Publishers Ltd.
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Current Analytical Chemistry, 2006, Vol. 2, No. 1
researchers harvest a large population of cells from an organism of interest to produce a homogenous mixture that is then subjected to analysis. An average in statistics is obtained from cells population. It is effective for cells that exhibit the same physiological properties. However, it becomes problematic when individual cells are in heterogenous conditions [24]. For example, the genotype and phenotype of cancer cells are quite heterogeneous [25, 26]. The result of bulk analysis can lead to oversimplification and the overlooking of vital processes that would otherwise go undetected. Increased sample handling associated with bulk analysis of cells often leads to sample bias [27] (Fig. (1)). Such bias can originate from enzymatic modifications occurring in bulk solution after lysis between an enzyme and substrate that are not normally co-localized. Thus, the importance of single cell examination is self-evident. Another example is the central nervous system. The function of a particular neuronal system is based on the properties and connections between heterogeneous populations of cells. The neuron cells are highly differentiated so that cell averages are particularly problematic and single cell measurement is required for detailed understanding. In the aspect of personalized medicine, in which every patient is treated with a unique combination of therapeutics tailored to combat disease, there is also an urgent demand on single cell approaches [28]. Only a small portion of the transcripts present in the cell determines the identity of the cell. These crucial transcripts are expressed at low levels and are often difficult to be detected with conventional approaches such as expression arrays. It is clear that to develop more efficient methods for SCP is vital. The term of SCP is originally proposed by Dovichi in 2000 [23]. SCP can be defined as protein identification and quantification within a single cell, and then followed by the determination of their localization, modifications, interactions, activities and functions. With the help of SCP, we can understand the regulation of proteins expressed at
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extreme levels, that is, single cell at single copy. And we may find a way to develop personalized medicine based on each patient’s own proteomics as well. 2. CLASSICAL APPROACHES IN PROTEOMICS Compared with genomics that is relatively static, homogeneous and easily amplified by PCR, proteomics shows more dynamic and heterogeneous characteristics. And there is no amplification method available for the sample. The classical approaches widely adopted in current proteomics include two-dimensional electrophoresis (2DE) for protein separation, mass spectrometry (MS) for protein identification, array-based chips and yeast two-hybrid experiments for protein interaction, x-ray diffraction and computer simulation for protein structure etc. [1]. 2DE is a multi-step procedure that can be used to separate hundreds to thousands of proteins with extremely high resolution. It works through separating proteins by their intrinsic isoelectric points in the first dimension (called isoelectric focusing), and then by their molecular weights in the second dimension. The advantages of 2DE are its tolerance to crude protein mixture, high sample load, and high resolution that more than 3000~4000 proteins can be resolved in a normal gel size. MS has increasingly become the predominant platform for the characterization of proteins. A mass spectrometer consists of an ion source, a mass analyzer that measures the mass-to-charge ratio (m/z) of the ionized analytes, and a detector that registers the number of ions at each m/z value. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the two key techniques most commonly used to volatize and ionize the proteins or peptides for MS analysis [29, 30]. The MS-based proteomics protocol is shown in Fig. (2). The proteins mixture are firstly separated by 2DE and then digested with enzymes into peptides that are softly ionized for further MS analyses. Each peptide is identified by bioinformatic approaches. While such approach showing great effectiveness in dealing with routine biological specimens, it does not in SCP. For example, the conventional 2DE commonly deals with large amounts of samples, which obviously is incompatible with a sample size at single cell level. Furthermore, it exhibits some disadvantages such as low reproducibility, low abundance protein discrimination, and high cost on human and financial resources. Therefore it is commented as more of ‘an art’ than ‘a science’. Another issue concerns MS. Though it does identify a great number of proteins, sensitivity needs to be further improved for low abundance proteins [31]. 3. CURRENT METHODS IN SCP RESEARCH 3.1 Capillary Electrophoresis
Fig. (1). Comparison of six constituents (numbered 1 through 6) of human colon cells (N=20) and bulk analysis (N=18) procedures using capillary electrophoresis. Peaks 4, 5, and 6 are significantly different (p
